RESEARCH ARTICLE
The immunomodulatory quinoline-3carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis Nina Franse´n Pettersson☯, Adnan Deronic☯, Julia Nilsson, Tine D. Hannibal, Lisbeth Hansen, Anja Schmidt-Christensen, Fredrik Ivars, Dan Holmberg* Department of Experimental Medical Science, Lund University, Lund, Sweden
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OPEN ACCESS Citation: Franse´n Pettersson N, Deronic A, Nilsson J, Hannibal TD, Hansen L, Schmidt-Christensen A, et al. (2018) The immunomodulatory quinoline-3carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis. PLoS ONE 13(9): e0203228. https://doi.org/ 10.1371/journal.pone.0203228
☯ These authors contributed equally to this work. *
[email protected]
Abstract Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages.
Editor: Matias A. Avila, University of Navarra School of Medicine and Center for Applied Medical Research (CIMA), SPAIN Received: June 25, 2018 Accepted: August 16, 2018
Introduction
Published: September 5, 2018
The quinoline-3-carboxamides (Q compounds) are small molecule compounds that have exhibited beneficial effects in several mouse models of inflammatory disease. One such compound, Paquinimod (ABR-215757), has been shown to ameliorate collagenase-induced osteoarthritis [1] as well as symptoms in mouse models of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) [2]. Phas also had beneficial effects in systemic lupus erythematosus (SLE) patients [3] and, more recently, in systemic sclerosis (SSc) patients [4]. Q compounds, such as Paquinimod have been reported to bind to the protein S100A9 [5], a proinflammatory mediator that also possesses chemotactic effects and is involved in the recruitment of cells of myeloid origin [6, 7]. The interaction of S100A9 with its receptors is inhibited by Paquinimod [5], and this inhibition has been suggested to underlie the anti-inflammatory effects of Paquinimod [2]. In this study, we evaluated the efficacy of Paquinimod in inhibiting the spontaneous, chronic liver inflammation and fibrosis that occur in the recently established NOD-Inflammation Fibrosis (N-IF) mouse model [8]. N-IF mice spontaneously develop chronic inflammation
Copyright: © 2018 Franse´n Pettersson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by (DH) NovoNordisk Fonden, NNF15OC0016146, Diabetesfonden, DIA2017-221, Barndiabetesfonden, and the Lundberg Foundation. The funders had no role in study design, data
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collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
and liver fibrosis driven by T-cell receptor (TCR) transgenic natural killer T (NKT)-II cells generated in immunodeficient NOD.Rag2-/- mice. Several components of the liver pathology in the N-IF mouse overlap with those of human conditions in which a progressive chronic inflammation precedes the development of fibrosis. Moreover, the fibrosis developing in the N-IF mouse liver has been demonstrated to be associated with an accumulation of α-SMA expressing hepatic stellate cells [8]. These phenotypic similarities make the N-IF mouse model unique compared to other available rodent models for liver fibrosis and provides a novel tool to test the efficacy of anti-fibrotic drug candidates. Using this model, we here show that Paquinimod has a therapeutic effect not only on the inflammation in the N-IF mouse liver but also significantly reduces the established liver fibrosis. This was accompanied with reduced numbers of TCR transgenic NKT-II cells, and their type 2-cytokine expression, as well as with reduced numbers of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages.
Material and methods Ethics statement Animal experiments were performed in strict accordance with the recommendations for the use of laboratory animals from the Swedish Board of Agriculture. The ethics committees of the local committee on the ethics of animal experiments of Malmo¨ and Lund permits no M143-11 and M52-16. Mice were sacrificed by cervical dislocation. All efforts were made to minimize suffering.
Animal studies N-IF (24αβNOD.Rag2-/-) and 24αβNOD control mice were bred and maintained in a specific pathogen-free facility at the Biomedical Center animal facility in Lund, Sweden. To study the effects of the Q compound Paquinimod, mice aged 8–9 weeks were treated with Paquinimod dissolved in drinking water corresponding to a daily dose of 25 mg/kg body weight/day for 4 or 10 weeks. Paquinimod was provided by Active Biotech, Lund, Sweden.
Histology Following 4 or 10 weeks of treatment, the mice were anesthetized and perfused with PBS via intra-cardiac puncture. Livers and spleens were weighed, and organ biopsies were fixed in 4% neutral-buffered formalin, embedded in paraffin and sectioned. Sections (5 μm) were stained with hematoxylin and eosin (H&E) and Masson’s trichrome and evaluated by microscopy.
Hydroxyproline measurement The hydroxyproline content of the liver and spleen was determined using the Hydroxyproline Colorimetric Assay Kit (BioVision, Milpitas, CA, USA) according to manufacturer’s instructions. Briefly, 100 μl of wet tissue (10 mg) was hydrolyzed in 100 μl of 12 N HCl for 3 h at 120˚C. Aliquots were transferred to a 96-well plate, dried overnight and incubated with chloramine T and DMAB reagents for 90 min at 60˚C. Finally, the absorbance at 560 nm was measured.
Gene expression analysis RNA was extracted from liver and spleen biopsies by the homogenization of tissues in QIAzol Lysis Reagent (Qiagen, Hilden, Germany), followed by purification on RNeasy Mini columns (Qiagen) according to the manufacturer’s protocol. RNA quality was evaluated using the A280/
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A260 ratio. One μg of total RNA was reverse transcribed using the RT2 HT First Strand Kit (Qiagen). Next, cDNA synthesis was performed in a C1000 Thermal Cycler (Bio-Rad, Hercules, CA) at 42˚C for 15 min, and the reaction was terminated at 95˚C for 5 min. The cDNA was subsequently used for quantitative PCR analysis using the RT2 SYBR Green MasterMix (Qiagen) at 95˚C for 5 min, followed by 40 cycles at 95˚C for 15 sec and 60˚C for 1 min in a MyiQ Thermal Cycler (Bio-Rad). The analyses were performed using primers for mouse CCR2, MMP2, MMP8 and TGF-β. GAPDH was used as a housekeeping gene. Fold changes in mRNA expression of the genes were calculated by using the ΔΔCt method. The relative normalized expression of each gene for each sample was calculated using the formula 2-ΔΔCt where the 24αβNOD mice treated with vehicle for 4 weeks is the control group.
Cell preparation Leukocytes from livers were obtained by incubating liver sections in a solution of 1 mg/ml collagenase XI (Sigma-Aldrich, St. Louis, MO) for 40 min at 37˚C, mincing the tissue through a 70-μm mesh and separating the leukocytes by centrifugation with a 50/30 Percoll gradient (GE Healthcare, Uppsala, Sweden). Single-cell suspensions from the spleen were prepared by disrupting the tissue through a 70-μm mesh.
Antibodies and flow cytometry Streptavidin-Brilliant Violet 605 and the following antibodies were purchased from Biolegend (Nordic Biosite, Ta¨by, Sweden): anti-c-kit-APC-Cy7 (2B8), anti-CD11b-Alexa700 (M1/70), anti-CD45-APC-Cy7 (30-F11), anti-CD115-APC (AFS98), anti-CD206-FITC (C068C2), antiF4/80-PE-Cy7 (BM8), and anti-Ly6G-Brilliant Violet 421 (1A8). The following antibodies were purchased from eBioscience (Nordic Biosite): anti-CD11c-PE-Cy7 (N418), antiFcεR1-APC (MAR-1), anti-Vα3.2-APC (RR3-16), and anti-Vβ9-eFluor450 (MR10-2). Ly6Cbiotin (AL-21) and SiglecF-PE (E50-2440) were purchased from BD Biosciences (Stockholm, Sweden), goat anti-S100A9 (mAb. Santa Cruz, sc-8115, clone M-19) was purchased from Santa Cruz Biotechnology, Dallas, Texas). Prior to surface staining, cells were incubated with the 2.4G2 (anti-CD16/CD32) antibody (BD Biosciences) to prevent nonspecific binding. The cells were then stained with the surface antibodies in FACS buffer (PBS supplemented with 3% FCS). Fixable Viability Dye-eFluor506 purchased from eBioscience was used to detect dead cells. For intracellular staining of CD206, the Foxp3/Transcription Factor Staining Kit from eBioscience was used according to the manufacturer’s protocol. The analysis of stained cells was performed using the LSRII flow cytometer (BD Biosciences) and FlowJo software (TreeStar, Ashland, OR). For gating strategy see S1 Fig.
Cell activation and cytokine analysis Single cells from the spleen or liver (105 per well) were cultured in complete medium [RPMI 1640 medium supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, 2.5% sodium bicarbonate (7.5% solution), 1 mM sodium pyruvate and 69 μM 1-thioglycerol] and activated with anti-CD3 (4 μg/ml, clone 154-2C11, BD Biosciences) antibody. The supernatants were collected after 24 h and were analyzed for cytokines using the mouse Th1/Th2/Th17/Th22 13-plex (eBioscience) according to the manufacturer’s instructions.
Statistical analysis The results are presented as the means and standard error of the mean (SEM). Differences between two groups were considered significant when P
